The references cited in the present application are not admitted to be prior art to the claimed invention.
Exposure to HCV results in an overt acute disease in a small percentage of cases, while in most instances the virus establishes a chronic infection causing liver inflammation and slowly progresses into liver failure and cirrhosis. (Iwarson, 1994. FEMS Microbiol. Rev. 14, 201-204.) Epidemiological surveys indicate HCV plays an important role in hepatocellular carcinoma pathogenesis. (Kew, 1994. FEMS Microbiol. Rev. 14, 211-220, Alter, 1995. Blood 85, 1681-1695.)
The HCV genome consists of a single strand RNA about 9.5 kb in length, encoding a precursor polyprotein about 3000 amino acids. (Choo et al., 1989. Science 244, 362-364, Choo et al., 1989. Science 244, 359-362, Takamizawa et al., 1991. J. Virol. 65, 1105-1113.) The HCV polyprotein contains the viral proteins in the order: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
Individual viral proteins are produced by proteolysis of the HCV polyprotein. Host cell proteases release the putative structural proteins C, E1, E2, and p7, and create the N-terminus of NS2 at amino acid 810. (Mizushima et al., 1994. J. Virol. 68, 2731-2734, Hijikata et al., 1993. Proc. Natl. Acad. Sci. USA 90, 10773-10777.)
The non-structural proteins NS3, NS4A, NS4B, NS5A and NS5B presumably form the virus replication machinery and are released from the polyprotein. A zinc-dependent protease associated with NS2 and the N-terminus of NS3 is responsible for cleavage between NS2 and NS3. (Grakoui et al., 1993. J. Virol. 67, 1385-1395, Hijikata et al., 1993. Proc. Natl. Acad. Sci. USA 90, 10773-10777.)
A distinct serine protease located in the N-terminal domain of NS3 is responsible for proteolytic cleavages at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions. (Barthenschlager et al., 1993. J. Virol. 67, 3835-3844, Grakoui et al., 1993. Proc. Natl. Acad. Sci. USA 90, 10583-10587, Tomei et al., 1993. J. Virol. 67, 4017-4026.) RNA stimulated NTPase and helicase activities are located in the C-terminal domain of NS3.
NS4A provides a cofactor for NS3 protease activity. (Failla et al., J. Virol. 1994. 68, 3753-3760, De Francesco et al., U.S. Pat. No. 5,739,002.) NS4A enhances NS-dependent cleavage at the different NS3 protease cleavage sites and is a required cofactor for cleavage at the NS3/4A and NS4B/NS5B junctions. (Gallinari et al., Biochemistry 38:5620-5632, 1999.) NS3 protease activity can be measured using peptides containing a region corresponding to a cleavage junction. (Steinkühler et al., International Publication WO 97/08304, published Mar. 6, 1997.) Peptides modified to contain an ester linkage at the NS3 cleavage site can also be used as a substrate to measure NS3 protease activity. (Steinkühler et al., International Publication WO 97/08304, published Mar. 6, 1997, Bianchi et al., Analytical Biochemistry 237:239-244, 1996.)
Different types of labels and assays have been employed to measure NS3 protease activity. Such assays include radiolabel assays, fluorometric assays, and fluorescence resonance energy transfer (FRET) assays. (Taliani et al., Analytical Biochemistry 240:60-67, 1996, Kakiuchi et al., Journal of Virological Methods 80:77-84, 1999, Gallinari et al., Biochemistry 38:5620-5632, 1999, Berdichevsky et al., Journal of Virological Methods 107:245-255, 2003.)